1. Field
The invention is related to thrombolytic therapy with the plasmin, specifically by local delivery of active plasmin proximal to or directly at the clot site. This therapeutic method is particularly applicable to the dissolution of clots wherever catheter directed delivery is feasible, particularly for example in distal limbs (e.g. arms and legs).
2. Background
Plasmin, the principle fibrinolytic enzyme in mammals, is a serine protease with trypsin-like specificity. It derives from the inactive precursor plasminogen which circulates in plasma at a concentration of xcx9c1.5 xcexcM. Plasminogen activators such as tPA or urokinase cleave a single-chain plasminogen molecule at the Arg560-Val561 peptide bond, producing active plasmin. The resulting two chains of plasmin are held together by two interchain disulphide bridges. The light chain (25 kDa) carries the catalytic center and is homologous to trypsin and other serine proteases. The heavy chain (60 kDa) consists of five very homologous triple-loop structures called kringles. Some kringles contain the so called lysine-binding sites which are responsible for plasminogen/plasmin interaction with fibrin, xc3xa12-antiplasmin and other proteins.
In spite of the success of thrombolytic drugs such as tissue plasminogen activator (tPA), streptokinase and urokinase, various thrombotic disorders, including myocardial infarction, occlusive stroke, deep venous thrombosis and peripheral arterial disease, remain a serious clinical problem. It appears that the currently available thrombolytic agents have several important limitations. At best, TIMI flow 3 within 90 min is obtained in approximately 50% of patients, acute coronary reocclusion occurs in roughly 10% of patients, coronary recanalization requires on average 45 minutes or more, intracerebral bleedings occurs in 0.3% to 0.7% of patients, and the residual mortality is at least 50% of that without thrombolytic treatment. Therefore, it is not surprising that research in the area of thrombolytics has focused on improvement of the existing plasminogen activators and finding new ones in an attempt to improve their fibrin specificity and increase plasma half-life to allow bolus administration.
The focus of development has been new plasminogen activators and with the exception of novel plasminogen activator (nPA), they all exhibit better fibrin specificity. They preserve the circulating levels of fibrinogen, plasminogen, xc3xa12-antiplasmin, Factors VIII and V. Nevertheless, based on the results of Phase II clinical trials with TNK-tPA and nPA, the improved safety profile of the new plasminogen activators has not translated into the better clinical outcome following thrombolytic therapy. The percentage of moderate and major bleeding episodes, including intracranial hemorrhage and stroke, were comparable with original tPA. The clogged arteries were not opened earlier and the rate of re-occlusions remains unchanged. It appears that the only benefit these activators have is the prolonged plasma half-life and the possibility of bolus administration.
An abstract on the method of treatment provided herein was published in BLOOD, Journal of the American Society of Hematology, Nov. 15, 1998, Abstract 3400. The abstract disclosed the potential of active Plasmin as a thrombolytic agent with the shift in modern fibronolytic treatment from systemic to local delivery.
U.S. Pat. No. 5,288,489 assigned to Orion Therapeutic Systems, Inc. discloses a fibrinolysis and fibrinogenolysis treatment which includes parenterally introducing into the body of a human patient human plasmin in fibrinolytically and fibrinogenolytically active form at a concentration and for a time sufficient to permit fibrinolytically and fibrinogenolytically active human plasmin to reach a concentration about the site of an intravascular clot sufficient to lyse the clot and/or to reduce circulating fibrinogen levels. This patent discloses the generation of plasmin from plasminogen just prior to its introduction into the body. The plasminogen is activated, or an plasmin inhibitor is removed, just prior to administration.
This invention relates to the discovery that thrombolytic therapy can be improved by the administration of active plasmin directly at or proximal to the site of a clot. Plasmin autodegrades and has not been available for therapeutic administration. This invention provides a method of preparing active plasmin for such direct administration. Plasmin is obtained by isolating plasminogen from Cohn Fraction II and II, activating the plasminogen obtained to plasmin and isolating the plasmin in an aqueous solution having a pH of less than about 4 to provide a stable formulation of plasmin. Plasminogen may be activated by using a plasminogen activator, such as urokinase, bound to an affinity resin column, preferably a urokinase-sepharose column. The active plasmin may then be captured on a benzamidine affinity resin, preferably a benzamidine-Sepharose column, and finally eluted with a low pH buffer. The eluted plasmin is formulated in an aqueous solution, acidified to a pH of less than about 4, preferably, about 3.7. The aqueous plasmin solution may be lyophilized and is substantially free of plasminogen activator. No extensive manipulation is required to administer this formulation. Proof is provided herein that this is a viable therapy compared to the current use of plasminogen activators that can cause extraneous bleeding along with dissolution of a clot.